Intracellular Viral RNA Levels Research Articles

Proteolytic cleavage and inactivation of the TRMT1 tRNA modification enzyme by SARS-CoV-2 main protease

Nonstructural protein 5 (Nsp5) is the main protease of SARS-CoV-2 that cleaves viral polyproteins into individual polypeptides necessary for viral replication. Here, we show that Nsp5 binds and cleaves human tRNA methyltransferase 1 (TRMT1), a host enzyme required for a prevalent post-transcriptional modification in tRNAs. Human cells infected with SARS-CoV-2 exhibit a decrease in TRMT1 protein levels and TRMT1-catalyzed tRNA modifications, consistent with TRMT1 cleavage and inactivation by Nsp5. Nsp5 cleaves TRMT1 at a specific position that matches the consensus sequence of SARS-CoV-2 polyprotein cleavage sites, and a single mutation within the sequence inhibits Nsp5-dependent proteolysis of TRMT1. The TRMT1 cleavage fragments exhibit altered RNA binding activity and are unable to rescue tRNA modification in TRMT1-deficient human cells. Compared to wild-type human cells, TRMT1-deficient human cells infected with SARS-CoV-2 exhibit reduced levels of intracellular viral RNA. These findings provide evidence that Nsp5-dependent cleavage of TRMT1 and perturbation of tRNA modification patterns contribute to the cellular pathogenesis of SARS-CoV-2 infection.

Proteolytic cleavage and inactivation of the TRMT1 tRNA modification enzyme by SARS-CoV-2 main protease.

Nonstructural protein 5 (Nsp5) is the main protease of SARS-CoV-2 that cleaves viral polyproteins into individual polypeptides necessary for viral replication. Here, we show that Nsp5 binds and cleaves human tRNA methyltransferase 1 (TRMT1), a host enzyme required for a prevalent post-transcriptional modification in tRNAs. Human cells infected with SARS-CoV-2 exhibit a decrease in TRMT1 protein levels and TRMT1-catalyzed tRNA modifications, consistent with TRMT1 cleavage and inactivation by Nsp5. Nsp5 cleaves TRMT1 at a specific position that matches the consensus sequence of SARS-CoV-2 polyprotein cleavage sites, and a single mutation within the sequence inhibits Nsp5-dependent proteolysis of TRMT1. The TRMT1 cleavage fragments exhibit altered RNA binding activity and are unable to rescue tRNA modification in TRMT1-deficient human cells. Compared to wild-type human cells, TRMT1-deficient human cells infected with SARS-CoV-2 exhibit reduced levels of intracellular viral RNA. These findings provide evidence that Nsp5-dependent cleavage of TRMT1 and perturbation of tRNA modification patterns contribute to the cellular pathogenesis of SARS-CoV-2 infection.

Enterovirus E infects bovine peripheral blood mononuclear cells. Implications for pathogenesis?

Enterovirus E (EV-E) is a common viral pathogen endemic in cattle worldwide. Little is known, however, about its potential interactions with bovine immune cells. The EV-E-permissiveness of bovine peripheral blood mononuclear cells (PBMCs) was evaluated. The infectious titres of extracellular virus were measured and the intracellular viral RNA levels were determined by reverse transcription quantitative PCR after cell inoculation. The effects of EV-E on cell viability and proliferative response were investigated with a methyl thiazolyl tetrazolium bromide reduction assay, the percentages of main lymphocyte subsets and oxidative burst activity of blood phagocytes were determined with flow cytometry, and pro-inflammatory cytokine secretion was measured with an ELISA. Enterovirus E productively infected bovine PBMCs. The highest infectious dose of EV-E decreased cell viability and T-cell proliferation. All of the tested doses of virus inhibited the proliferation of high responding to lipopolysaccharide B cells and stimulated the secretion of interleukin 1β, interleukin 6 and tumour necrosis factor α pro-inflammatory cytokines. Interactions of EV-E with bovine immune cells may indicate potential evasion mechanisms of the virus. There is also a risk that an infection with this virus can predispose the organism to secondary infections, especially bacterial ones.

Homoseongomycin, a compound isolated from marine actinomycete bacteria K3-1, is a potent inhibitor of encephalitic alphaviruses

Marine microorganisms have been a resource for novel therapeutic drugs for decades. In addition to anticancer drugs, the drug acyclovir, derived from a marine sponge, is FDA-approved for the treatment of human herpes simplex virus-1 infections. Most alphaviruses that are infectious to terrestrial animals and humans, such as Venezuelan and eastern equine encephalitis viruses (VEEV and EEEV), lack efficient antiviral drugs and it is imperative to develop these remedies. To push the discovery and development of anti-alphavirus compounds forward, this study aimed to isolate and screen for potential antiviral compounds from cultured marine microbes originating from the marine environment. Compounds from marine microbes were of interest as they are prolific producers of bioactive compounds across the spectrum of human diseases and infections. Homoseongomycin, an actinobacteria isolated from a marine sponge displayed impressive activity against VEEV from a total of 76 marine bioactive products. The 50% effective concentration (EC50) for homoseongomycin was 8.6 μM for suppressing VEEV TC-83 luciferase reporter virus replication. Homoseongomycin was non-toxic up to 50 μM and partially rescued cells from VEEV induced cell death. Homoseongomycin exhibited highly efficient antiviral activity with a reduction of VEEV infectious titers by 8 log10 at 50 μM. It also inhibited EEEV replication with an EC50 of 1.2 μM. Mechanism of action studies suggest that homoseongomycin affects both early and late stages of the viral life cycle. Cells treated with 25 μM of homoseongomycin had a ~90% reduction in viral entry. In comparison, later stages showed a more robust reduction in infectious titers (6 log10) and VEEV extracellular viral RNA levels (4 log10), but a lesser impact on intracellular viral RNA levels (1.5 log10). In sum, this work demonstrates that homoseongomycin is a potential anti-VEEV and anti-EEEV compound due to its low cytotoxicity and potent antiviral activity.

Model-based approach for predicting the impact of genetic modifications on product yield in biopharmaceutical manufacturing-Application to influenza vaccine production.

A large group of biopharmaceuticals is produced in cell lines. The yield of such products can be increased by genetic engineering of the corresponding cell lines. The prediction of promising genetic modifications by mathematical modeling is a valuable tool to facilitate experimental screening. Besides information on the intracellular kinetics and genetic modifications the mathematical model has to account for ubiquitous cell-to-cell variability. In this contribution, we establish a novel model-based methodology for influenza vaccine production in cell lines with overexpressed genes. The manipulation of the expression level of genes coding for host cell factors relevant for virus replication is achieved by lentiviral transduction. Since lentiviral transduction causes increased cell-to-cell variability due to different copy numbers and integration sites of the gene constructs we use a population balance modeling approach to account for this heterogeneity in terms of intracellular viral components and distributed kinetic parameters. The latter are estimated from experimental data of intracellular viral RNA levels and virus titers of infection experiments using cells overexpressing a single host cell gene. For experiments with cells overexpressing multiple host cell genes, only final virus titers were measured and thus, no direct estimation of the parameter distributions was possible. Instead, we evaluate four different computational strategies to infer these from single gene parameter sets. Finally, the best computational strategy is used to predict the most promising candidates for future modifications that show the highest potential for an increased virus yield in a combinatorial study. As expected, there is a trend to higher yields the more modifications are included.

Visualization of cell-type dependent effects of anti-E2 antibody and interferon-gamma treatments on localization and expression of Broccoli aptamer-tagged alphavirus RNAs

Sindbis virus (SINV) is an alphavirus that causes age-dependent encephalomyelitis in mice. Within 7–8 days after infection infectious virus is cleared from neurons through the antiviral effects of antibody and interferon-gamma (IFNγ), but RNA persists. To better understand changes in viral RNA associated with immune-mediated clearance we developed recombinant strains of SINV that have genomic and subgenomic viral RNAs tagged with the Broccoli RNA aptamer that binds and activates a conditional fluorophore for live cell imaging of RNA. Treatment of SINV-Broccoli-infected cells with antibody to the SINV E2 glycoprotein had cell type-specific effects. In BHK cells, antibody increased levels of intracellular viral RNA and changed the primary location of genomic RNA from the perinuclear region to the plasma membrane without improving cell viability. In undifferentiated and differentiated AP7 (dAP7) neuronal cells, antibody treatment decreased levels of viral RNA. Occasional dAP7 cells escaped antibody-mediated clearance by not expressing cell surface E2 or binding antibody to the plasma membrane. IFNγ decreased viral RNA levels only in dAP7 cells and synergized with antibody for RNA clearance and improved cell survival. Therefore, analysis of aptamer-tagged SINV RNAs identified cell type- and neuronal maturation-dependent responses to immune mediators of virus clearance.

Establishment of a Cell Culture Model of Persistent Flaviviral Infection: Usutu Virus Shows Sustained Replication during Passages and Resistance to Extinction by Antiviral Nucleosides.

Chronic viral disease constitutes a major global health problem, with several hundred million people affected and an associated elevated number of deaths. An increasing number of disorders caused by human flaviviruses are related to their capacity to establish a persistent infection. Here we show that Usutu virus (USUV), an emerging zoonotic flavivirus linked to sporadic neurologic disease in humans, can establish a persistent infection in cell culture. Two independent lineages of Vero cells surviving USUV lytic infection were cultured over 82 days (41 cell transfers) without any apparent cytopathology crisis associated. We found elevated titers in the supernatant of these cells, with modest fluctuations during passages but no overall tendency towards increased or decreased infectivity. In addition to full-length genomes, viral RNA isolated from these cells at passage 40 revealed the presence of defective genomes, containing different deletions at the 5’ end. These truncated transcripts were all predicted to encode shorter polyprotein products lacking membrane and envelope structural proteins, and most of non-structural protein 1. Treatment with different broad-range antiviral nucleosides revealed that USUV is sensitive to these compounds in the context of a persistent infection, in agreement with previous observations during lytic infections. The exposure of infected cells to prolonged treatment (10 days) with favipiravir and/or ribavirin resulted in the complete clearance of infectivity in the cellular supernatants (decrease of ~5 log10 in virus titers and RNA levels), although modest changes in intracellular viral RNA levels were recorded (<2 log10 decrease). Drug withdrawal after treatment day 10 resulted in a relapse in virus titers. These results encourage the use of persistently-infected cultures as a surrogate system in the identification of improved antivirals against flaviviral chronic disease.

The retinoic acid receptor (RAR) α-specific agonist Am80 (tamibarotene) and other RAR agonists potently inhibit hepatitis B virus transcription from cccDNA

Chronic infection with the human Hepatitis B virus (HBV) is a major global health problem. Hepatitis D virus (HDV) is a satellite of HBV that uses HBV envelope proteins for cell egress and entry. Using infection systems encoding the HBV/HDV receptor human sodium taurocholate co-transporting polypeptide (NTCP), we screened 1181 FDA-approved drugs applying markers for interference for HBV and HDV infection. As one primary hit we identified Acitretin, a retinoid, as an inhibitor of HBV replication and HDV release. Based on this, other retinoic acid receptor (RAR) agonists with different specificities were found to interfere with HBV replication, verifying that the retinoic acid receptor pathway regulates replication. Of the eight agonists investigated, RARα-specific agonist Am80 (tamibarotene) was most active. Am80 reduced secretion of HBeAg and HBsAg with IC50s < 10 nM in differentiated HepaRG-NTCP cells. Similar effects were observed in primary human hepatocytes. In HepG2-NTCP cells, profound Am80-mediated inhibition required prolonged treatment of up to 35 days. Am80 treatment of cells with an established HBV cccDNA pool resulted in a reduction of secreted HBsAg and HBeAg, which correlated with reduced intracellular viral RNA levels, but not cccDNA copy numbers. The effect lasted for >12 days after removal of the drug. HBV genotypes B, D, and E were equally inhibited. By contrast, Am80 did not affect HBV replication in transfected cells or HepG2.2.15 cells, which carry an integrated HBV genome. In conclusion, our results indicate a persistent inhibition of HBV transcription by Am80, which might be used for drug repositioning.

Profiling of rotavirus 3′UTR-binding proteins reveals the ATP synthase subunit ATP5B as a host factor that supports late-stage virus replication

Genome replication and virion assembly of segmented RNA viruses are highly coordinated events, tightly regulated by sequence and structural elements in the UTRs of viral RNA. This process is poorly defined and likely requires the participation of host proteins in concert with viral proteins. In this study, we employed a proteomics-based approach, named RNA-protein interaction detection (RaPID), to comprehensively screen for host proteins that bind to a conserved motif within the rotavirus (RV) 3' terminus. Using this assay, we identified ATP5B, a core subunit of the mitochondrial ATP synthase, as having high affinity to the RV 3'UTR consensus sequences. During RV infection, ATP5B bound to the RV 3'UTR and co-localized with viral RNA and viroplasm. Functionally, siRNA-mediated genetic depletion of ATP5B or other ATP synthase subunits such as ATP5A1 and ATP5O reduced the production of infectious viral progeny without significant alteration of intracellular viral RNA levels or RNA translation. Chemical inhibition of ATP synthase diminished RV yield in both conventional cell culture and in human intestinal enteroids, indicating that ATP5B positively regulates late-stage RV maturation in primary intestinal epithelial cells. Collectively, our results shed light on the role of host proteins in RV genome assembly and particle formation and identify ATP5B as a novel pro-RV RNA-binding protein, contributing to our understanding of how host ATP synthases may galvanize virus growth and pathogenesis.

In Vitro Anti-hepatitis B Virus Activity of 2',3'-Dideoxyguanosine.

2',3'-dideoxyguanosine (DoG) has been demonstrated to inhibit duck hepatitis B virus (DHBV) replication in vivo in a duck model of HBV infection. In the current study, the in vitro antiviral effects of DoG on human and animal hepadnaviruses were investigated. Our results showed that DoG effectively inhibited HBV, DHBV, and woodchuck hepatitis virus (WHV) replication in hepatocyte-derived cells in a dose-dependent manner, with 50% effective concentrations (EC50) of 0.3 ± 0.05, 6.82 ± 0.25, and 23.0 ± 1.5μmol/L, respectively. Similar to other hepadnaviral DNA polymerase inhibitors, DoG did not alter the levels of intracellular viral RNA but induced the accumulation of a less-than-full-length viral RNA species, which was recently demonstrated to be generated by RNase H cleavage of pgRNA. Furthermore, using a transient transfection assay, DoG showed similar antiviral activity against HBV wild-type, 3TC-resistant rtA181V, and adefovir-resistant rtN236T mutants. Our results suggest that DoG has potential as a nucleoside analogue drug with anti-HBV activity.

Serum-derived IgG from coxsackievirus A6-infected patients can enhance the infection of peripheral blood mononuclear cells with coxsackievirus A6

Coxsackievirus A6 (CV-A6) has recently emerged as an enterovirus causing Hand Foot and Mouth Disease with severe complications. The pathogenic mechanisms of CV-A6- associated Hand foot and Mouth disease are largely unknown. In this study, it was investigated whether serum and IgG from patients with CV-A6 infection can enhance the infection of PBMC with the virus. Serum samples were obtained from five children with CV-A6 infection confirmed by RT-PCR and seven controls. IgG was isolated from serum by using affinity chromatography columns. CV-A6 was incubated with serum or IgG from controls and patients then the mixtures were added to PBMC cultures. The levels of IFNα in supernatants were measured by ELISA, and the levels of intracellular viral RNA were measured by RT-qPCR. It has been observed that there is an anti-CV-A6 enhancing activity in serum and serum-derived immunoglobulin G of children with CV-A6 infection but not in those of uninfected controls. Whether this activity has implications in the pathogenesis of CV-A6 associated diseases should be investigated.

Establishment of an antiviral assay system and identification of severe fever with thrombocytopenia syndrome virus inhibitors.

AimsSevere fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne infectious disease. SFTS is epidemic in Asia, and its fatality rate is around 30% in Japan. The causative virus severe fever with thrombocytopenia syndrome virus (SFTSV) is a phlebovirus of the family Phenuiviridae (the order Bunyavirales). Although effective treatments are required, there are no antiviral agents currently approved for clinical use. Ribavirin and favipiravir were examined for their anti-SFTSV activity and found to be selective inhibitors of SFTSV replication in vitro. However, their activity was not sufficient. Therefore, it is mandatory to identify novel compounds active against SFTSV. To this end, we have established a safe and rapid assay system for screening selective inhibitors of SFTSV.MethodsThe virus was isolated from SFTS patients treated in Kagoshima University Hospital. Vero cells were infected with SFTSV and incubated in the presence of various concentrations of test compounds. After three days, the cells were examined for their intracellular viral RNA levels by real-time reverse transcription-PCR without extracting viral RNA. The cytotoxicity of test compounds was determined by a tetrazolium dye method.ResultsAmong the test compounds, the antimalarial agent amodiaquine was identified as a selective inhibitor of SFTSV replication. Its 50% effective concentration (EC50) and cytotoxic concentration (CC50) were 19.1 ± 5.1 and >100 µM, respectively. The EC50 value of amodiaquine was comparable to those of ribavirin and favipiravir.ConclusionAmodiaquine is considered to be a promising lead of novel anti-SFTSV agents, and evaluating the anti-SFTSV activity of its derivatives is in progress.

GRIM-19 Restricts HCV Replication by Attenuating Intracellular Lipid Accumulation

Gene-associated with retinoid-interferon-induced mortality 19 (GRIM-19) targets multiple signaling pathways involved in cell death and growth. However, the role of GRIM-19 in the pathogenesis of hepatitis virus infections remains unexplored. Here, we investigated the restrictive effects of GRIM-19 on the replication of hepatitis C virus (HCV). We found that GRIM-19 protein levels were reduced in HCV-infected Huh7 cells and Huh7 cells harboring HCV replicons. Moreover, ectopically expressed GRIM-19 caused a reduction in both intracellular viral RNA levels and secreted viruses in HCVcc-infected cell cultures. The restrictive effect on HCV replication was restored by treatment with siRNA against GRIM-19. Interestingly, GRIM-19 overexpression did not alter the level of phosphorylated STAT3 or its subcellular distribution. Strikingly, forced expression of GRIM-19 attenuated an increase in intracellular lipid droplets after oleic acid (OA) treatment or HCVcc infection. GRIM-19 overexpression abrogated fatty acid-induced upregulation of sterol regulatory element-binding transcription factor-1 (SREBP-1c), resulting in attenuated expression of its target genes such as fatty acid synthase (FAS) and acetyl CoA carboxylase (ACC). Treatment with OA or overexpression of SREBP-1c in GRIM-19-expressing, HCVcc-infected cells restored HCV replication. Our results suggest that GRIM-19 interferes with HCV replication by attenuating intracellular lipid accumulation and therefore is an anti-viral host factor that could be a promising target for HCV treatment.

Live attenuated influenza vaccine strains elicit a greater innate immune response than antigenically-matched seasonal influenza viruses during infection of human nasal epithelial cell cultures

Influenza viruses are global pathogens that infect approximately 10–20% of the world's population each year. Vaccines, including the live attenuated influenza vaccine (LAIV), are the best defense against influenza infections. The LAIV is a novel vaccine that actively replicates in the human nasal epithelium and elicits both mucosal and systemic protective immune responses. The differences in replication and innate immune responses following infection of human nasal epithelium with influenza seasonal wild type (WT) and LAIV viruses remain unknown. Using a model of primary differentiated human nasal epithelial cell (hNECs) cultures, we compared influenza WT and antigenically-matched cold adapted (CA) LAIV virus replication and the subsequent innate immune response including host cellular pattern recognition protein expression, host innate immune gene expression, secreted pro-inflammatory cytokine production, and intracellular viral RNA levels. Growth curves comparing virus replication between WT and LAIV strains revealed significantly less infectious virus production during LAIV compared with WT infection. Despite this disparity in infectious virus production the LAIV strains elicited a more robust innate immune response with increased expression of RIG-I, TLR-3, IFNβ, STAT-1, IRF-7, MxA, and IP-10. There were no differences in cytotoxicity between hNEC cultures infected with WT and LAIV strains as measured by basolateral levels of LDH. Elevated levels of intracellular viral RNA during LAIV as compared with WT virus infection of hNEC cultures at 33°C may explain the augmented innate immune response via the up-regulation of pattern recognition receptors and down-stream type I IFN expression. Taken together our results suggest that the decreased replication of LAIV strains in human nasal epithelial cells is associated with a robust innate immune response that differs from infection with seasonal influenza viruses, limits LAIV shedding and plays a role in the silent clinical phenotype seen in human LAIV inoculation.

Evaluation of the inactivation of human Coxsackievirus by thermophilic and mesophilic anaerobic digestion using integrated cell culture and reverse transcription real-time quantitative PCR

The virucidal effects of anaerobic digestion were evaluated using human Coxsackievirus as a model for the Enterovirus family. Coxsackievirus was inactivated completely by thermophilic anaerobic digestion (TAD). By 4 h no living and infectious virus remained and no detectable viral RNA was present after 2 days in TAD (7.0 log reduction). Compared to TAD, 2.6 log reduction of viral RNA was achieved by 14 days in mesophilic anaerobic digestion (MAD) (p < 0.0001). Although cytopathogenic effect was not observed in the cultured cells, low levels of intracellular viral RNA were detected after one day of MAD treatment indicating that Coxsackievirus had infected the cells but could not replicate. The combination of thermal and biochemical effects in TAD plays a critical role for viral disinfection. The results of this study indicate that selection of the right configuration of anaerobic digestion for treatment of biowaste may reduce the risk of viral contamination to the environment and water source.

Sindbis virus induced phosphorylation of IRF3 in human embryonic kidney cells is not dependent on mTOR

The mammalian target of rapamycin (mTOR) plays critical roles in immunity. We previously showed that infection of human embryonic kidney (HEK) cells with Sindbis virus (SIN), an enveloped RNA alphavirus, profoundly suppresses Akt/mTOR signaling, and host translation late during infection. To understand how SIN mediated suppression of mTOR affects innate response, we analyzed phosphorylation of interferon regulatory factor 3 (IRF3) and expression of two antiviral genes. Here we show strong phosphorylation of IRF3, and an increase in mRNA levels for antiviral genes interferon stimulated gene (ISG)56 and interferon gamma inducible protein (IP)-10 when intracellular viral RNA levels are high during late infection. The mTOR inhibitors rapamycin and torin1 do not block, but mildly upregulate these responses. Even after prolonged treatment with Ly294002, the PI3K inhibitor only partially blocks SIN induced phosphorylation of IRF3. While Ly294002 treatment downregulated the SIN induced expression of ISG56 mRNA levels, it had no effect on SIN induced upregulation of IP-10 expression. These results point to SIN replication-mediated activation of IRF3, independent of mTOR function, when host protein synthesis is severely suppressed by virus infection.

Medicinal herbal extracts of Sophorae radix Acanthopanacis cortex Sanguisorbae radix and Torilis fructus inhibit coronavirus replication in vitro

Cimicifuga rhizome, Meliae cortex, Coptidis rhizome and Phellodendron cortex have been previously shown to exhibit anti-coronavirus activity. Here, an additional 19 traditional medicinal herbal extracts were evaluated for antiviral activities in vitro. A plaque assay was used to evaluate the effects of 19 extracts, and the concentration of extract required to inhibit 50% of the replication (EC(50)) of mouse hepatitis virus (MHV) A59 strain (MHV-A59) was determined. The 50% cytotoxic concentration (CC(50)) of each extract was also determined. Northern and western blot analyses were conducted to evaluate antiviral activity on viral entry, viral RNA and protein expression, and release in MHV-infected DBT cells. Sophorae radix, Acanthopanacis cortex and Torilis fructus reduced intracellular viral RNA levels with comparable reductions in viral proteins and MHV-A59 production. The extracts also reduced the replication of the John Howard Mueller strain of MHV, porcine epidemic diarrhoea virus and vesicular stomatitis virus in vitro. Sanguisorbae radix reduced coronavirus production, partly as a result of decreased protein synthesis, but without a significant reduction in intracellular viral RNA levels. The EC(50) values of the four extracts ranged from 0.8 to 3.7 microg/ml, whereas the CC(50) values ranged from 156.5 to 556.8 microg/ml. Acanthopanacis cortex and Torilis fructus might exert their antiviral activities in MHV-A59-infected cells by inducing cyclooxygenase-2 expression via the activation of extracellular signal-related kinase (ERK) and p38 or ERK alone, respectively. Sophorae radix, Acanthopanacis cortex, Sanguisorbae radix and Torilis fructus might be considered as promising novel anti-coronavirus drug candidates.

An interfering activity against lymphocytic choriomeningitis virus replication associated with enhanced mutagenesis

Previous studies have documented that, in the presence of the mutagenic base analogue 5-fluorouracil (FU), lymphocytic choriomeningitis virus (LCMV) that persisted in BHK-21 cells decreased its infectivity to a larger extent than intracellular viral RNA levels, prior to virus extinction. This observation, together with in silico simulations, led to the proposal of the lethal defection model of virus extinction. This model suggests the participation of defective-interfering genomes in the loss of infectivity by increased mutagenesis. Since LCMV naturally produces defective-interfering particles, it was important to show that a capacity to interfere is produced in association with FU treatment. Here, we document that BHK-21 cells persistently infected with LCMV grown in the presence of FU, but not in its absence, generated an interfering activity that suppressed LCMV infectivity. Interference was specific for LCMV and was sensitive to UV irradiation and its activity was dose- and time-dependent. The interfering preparations produced positive LCMV immunofluorescence and viral particles seen by electron microscopy when used to infect cells, despite some preparations being devoid of detectable infectivity. Interference did not involve significant increases of mutant spectrum complexity, as predicted by the lethal defection model. The results provide support for a specific interference associated with LCMV when the virus replicates in the presence of FU. The excess of interference relative to that observed in the absence of FU is necessary for LCMV extinction.

Suppression of hepatitis C virus replication by protein kinase C‐related kinase 2 inhibitors that block phosphorylation of viral RNA polymerase

Hepatitis C virus (HCV) infection is a serious threat to human health worldwide. In spite of the continued search for specific and effective anti-HCV therapies, the rapid emergence of drug-resistance variants has been hampering the development of anti-HCV drugs designed to target viral enzymes. Targeting host factors has therefore emerged as an alternative strategy offering the potential to circumvent the ever-present complication of drug resistance. We previously identified protein kinase C-related kinase 2 (PRK2) as a cellular kinase that phosphorylates the HCV RNA-dependent RNA polymerase (RdRp). Here, we report the anti-HCV activity of HA1077, also known as fasudil, and Y27632, which blocks HCV RdRp phosphorylation by suppressing PRK2 activation. Treatment of a Huh7 cell line, stably expressing a genotype 1b HCV subgenomic replicon RNA, with 20 microm each of HA1077 and Y27632 reduced the HCV RNA level by 55% and 30%, respectively. A combination of the inhibitors with 100 IU/mL interferon alpha (IFN-alpha) significantly potentiated the anti-HCV drug activities resulting in approximately a 2-log(10) viral RNA reduction. We also found that IFN-alpha does not activate PRK2 as well as its upstream kinase PDK1 in HCV-replicating cells. Furthermore, treatment of HCV-infected cells with 20 microm each of HA1077 and Y27632 reduced the levels of intracellular viral RNA by 70% and 92%, respectively. Taken together, the results identify PRK2 inhibitors as potential antiviral drugs that act by suppressing HCV replication via inhibition of viral RNA polymerase phosphorylation.

Dynein-Dependent Transport of the Hantaan Virus Nucleocapsid Protein to the Endoplasmic Reticulum-Golgi Intermediate Compartment

In contrast to most negative-stranded RNA viruses, hantaviruses and other viruses in the family Bunyaviridae mature intracellularly, deriving the virion envelope from the endoplasmic reticulum (ER) or Golgi compartment. While it is generally accepted that Old World hantaviruses assemble and bud into the Golgi compartment, some studies with New World hantaviruses have raised the possibility of maturation at the plasma membrane as well. Overall, the steps leading to virion assembly remain largely undetermined for hantaviruses. Because hantaviruses do not have matrix proteins, the nucleocapsid protein (N) has been proposed to play a key role in assembly. Herein, we examine the intracellular trafficking and morphogenesis of the prototype Old World hantavirus, Hantaan virus (HTNV). Using confocal microscopy, we show that N colocalized with the ER-Golgi intermediate compartment (ERGIC) in HTNV-infected Vero E6 cells, not with the ER, Golgi compartment, or early endosomes. Brefeldin A, which effectively disperses the ER, the ERGIC, and Golgi membranes, redistributed N with the ERGIC, implicating membrane association; however, subcellular fractionation experiments showed the majority of N in particulate fractions. Confocal microscopy revealed that N was juxtaposed to and distributed along microtubules and, over time, became surrounded by vimentin cages. To probe cytoskeletal association further, we probed trafficking of N in cells treated with nocodazole and cytochalasin D, which depolymerize microtubules and actin, respectively. We show that nocodazole, but not cytochalasin D, affected the distribution of N and reduced levels of intracellular viral RNA. These results suggested the involvement of microtubules in trafficking of N, whose movement could occur via molecular motors such as dynein. Overexpression of dynamitin, which is associated with dynein-mediated transport, creates a dominant-negative phenotype blocking transport on microtubules. Overexpression of dynamitin reduced N accumulation in the perinuclear region, which further supports microtubule components in N trafficking. The combined results of these experiments support targeting of N to the ERGIC prior to its movement to the Golgi compartment and the requirement of an intact ERGIC for viral replication and, thus, the possibility of virus factories in this region.